The present invention relates to syntheses of the human T-antigenic determinant and antigens and immunoabsorbents formed therefrom.
Over fifty years ago, Thomsen.sup.1 observed that human red cells, in vitro, could become transformed such that the cells became agglutinated by normal ABO compatible sera. After extensive investigation, Friedenreich.sup.2 concluded that Thomsen's agent was a bacterial enzyme which degraded a natural antigenic determinant to liberate the so-called T receptor--a structure which is bound by an agglutinin of general occurrence in human sera. It is now established that the enzyme responsible for the transformation is a neuraminidase which exposed the T determinant by removing N-acetyl-.alpha.-D-neuraminidic acid residues (.alpha.-sialosides) from certain sialoglycoproteins.sup.3.
In 1966, the structure of this determinant was shown by Kim and Uhlenbruck.sup.4 to be the disaccharide BDGal(1.fwdarw.3).alpha.DGalNAc which, in the glycoprotein, is glycosidically linked to a threonine or serine residue. The T-determinant is now known to occur in a wide variety of glycoproteins.sup.5,6.
Recent findings that this structure occurs in tumor-associated antigens has caused a resurgence of interest in the investigation of this antigen and the corresponding antibodies. In particular, various investigators have shown that the T antigen can be demonstrated on tumor cells of animal and human origin.sup.7-10 ; that immediate and delayed type hypersensitivity reactions to the T antigen can be demonstrated in patients with certain forms of cancer.sup.11 ; and that changes in serum anti-T levels can be of diagnostic significance with regard to some cancers.sup.11-12 .
All of these observations have obvious important clinical implications both potential and realized. However, one major difficulty in developing the clinical applications is that prior to the present invention, the key component, a T antigen, was only available from natural sources chiefly through the laborious extraction of enzymatically treated human erythrocyte membranes.sup.13 . This procedure affords only relatively small amounts of material which, because of its origin, is inherently difficult to purify and characterize. In addition, for applications such as delayed hypersensitivity testing in which T antigenic material is injected into humans, there is the added disadvantage that materials such as this, derived from human blood products, carry the risk of transmission of hepatitis.
The synthesis of a compound, O-.beta.-D-galactopyranosyl-(1.fwdarw.3)-O-(2-acetamido-2-deoxy-.alpha.-D- galactopyranosyl)-N-Tosyl-L-serine, which contains the terminal disaccharide of the T-antigenic determinant, has been reported.sup.14 . However, this compound was never demonstrated to have utility as the human T-antigenic determinant. It is not readily apparent whether the compound could be linked to an antigen-forming carrier molecule and further whether the resulting conjugate, if formed, would function as an artificial T-antigen. In fact it would be predicted that the unnatural highly antigenic N-tosyl group present in the aglycone moiety would cause antigens from this compound to be immunochemically dissimilar from the natural T-antigen.
In U.S. Pat. No. 4,137,401 issued to Lemieux, Bundle and Baker, a bridging arm is disclosed O-.beta.-glycosidically linked to aldose moieties. The bridging arm has the structure O-R-COR" where R is an aliphatic hydrocarbon moiety having 3-17 carbon atoms and R" is H, OH, NH.sub.2 , NHNH.sub.2, N.sub.3 or a lower alkoxy group. The bridging arm enables one to link carbohydrate antigenic determinants to carrier molecules or solid supports to produce artificial antigens and immunoabosrbents. It should be appreciated that the reaction conditions set forth in the reference for attaching the bridging arm to the aldose moiety are those which will produce a .beta.-D-anomeric glycosidic linkage. An .alpha.-D-anomeric bridging arm is desired in the T-antigenic determinant.
In U.S. Pat. No. 4,195,174 issued Mar. 25, 1980, to Lemieux and Ratcliffe, processes are provided for the syntheses of O-acylated-2-azido-2-deoxy glycosyl halides. These halides can be converted to O-acylated-2-azido-2-deoxy glycosides. In particular the patent reports the synthesis of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.alpha.-D-galactopyranosyl chloride and its reaction with alcohols, including an aglycone bridging arm, to form 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.alpha.-D-galactopyranosides.